This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Clostridial neurotoxins (CNTs), such as botulinum (BoNT) and tetanus (TeNT) neurotoxins, are the causative agents of the neuroparalytic diseases tetanus and botulism. CNTs impair neuronal exocytosis by specific proteolysis of SNARE proteins once inside the neuron, resulting in the clinical manifestations of flaccid and spastic motor paralysis. CNTs bind with high specificity at neuromuscular junctions. The molecular details of the toxin-cell recognition have been elusive. We reported the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15-[unreadable] resolution. On binding, a helix is induced in the luminal domain that binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. The mechanism by which a CNT properly identifies and cleaves its target SNARE once inside the neuron involves one or more regions of enzyme-substrate interaction remote from the active site (exosites). Our studies provide the basis for the development of preventive vaccines or inhibitors against these neurotoxins for bio defense, as well as design of modified neurotoxins with different target specificities for clinical applications. In addition, this work is a paradigm for protease inhibitor development in general since proteases represent major challenges for drug development due to the inherent flexibility of these enzymes.